Initiation of protein synthesis is highly regulated and is the endpoint of cellular signaling pathways;misfunction of initiation is strongly linked to cancer. Initiation involves many host factors that assist ribosomal subunit binding to the 5'end of mRNAs;subsequently, this ribosomal complex directionally scans to the first start codon, where an 80S complex assembles, poised to start protein synthesis. Despite increasing structural knowledge of the components, a full mechanistic and dynamic understanding of translation initiation and its regulation remains elusive. Here, we harness biochemical approaches to apply single-molecule fluorescence methods to eukaryotic translation initiation. In specific aim 1, single-molecule fluorescence will be used to determine the timing and order of initiation factor and 40S subunit binding to a model mRNA that allows initiation without scanning. In collaboration with J. Lorsch (JHU), we will use dye-labeled yeast translation factors. In specific aim 2, we will study later steps in initiation, such as GTP hydrolysis and 60S subunit joining. We will determine the timing and dynamics of subunit joining and the role of GTP hydrolysis in these steps. We will use antibiotics and toxins to delineate mechanistic steps in initiation. In specific aim 3, we will build further complexity into the system by adding 5'UTR scanning on a real mRNA 5'UTR. We will observe FRET between ribosomal subunit and mRNA to monitor movements of the initiation complex to the start codon. We will test the role of mRNA structure in the 5'UTR on scanning dynamics. In specific aim 4, we will probe the nature and duration of proposed 5'- to 3'- end circularization in modulating initiation efficiency. We will couple these observations to those of protein production through detection of dye-labeled protein on the translating ribosome. The results of this proposal will provide a biophysical context to understanding eukaryotic translation, its regulation, and role in human disease.